Yeast organisms produce a number of proteins which are synthesized intracellularly, but which have a function outside the cell. Such extracellular proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-protein form containing a presequence ensuring effective direction of the expressed product across the membrane of the endoplasmic reticulum (ER). The presequence, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeifer, S. R. and Rothman, J. E. Ann. Rev. Biochem. 56 (1987) 829-852).
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast. European published patent application No. 88 632 describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression vehicle harbouring DNA encoding the desired protein and a signal peptide, preparing a culture of the transformed organism, growing the culture and recovering the protein from the culture medium. The signal peptide may be the signal peptide of the desired protein itseft, a heterologous signal peptide or a hybrid of native and heterologous signal peptide.
A problem encountered with the use of signal peptides heterologous to yeast might be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage after the signal peptide.
The Saccharomyces cerevisiae MF.alpha.I (.alpha.-factor) is synthesized as a prepro form of 165 amino acids comprising signal- or prepeptide of 19 amino acids followed by a "leader" or propeptide of 64 amino acids, encompassing three N-linked glycosylation sites followed by (LysArg((Asp/Glu)Ala).sub.2-3 .alpha.-factor).sub.4 (Kurjan, J. and Herskowitz, I. Cell 30 (1982) 933-943). The signal-leader part of the preproMF.alpha.1 has been widely employed to obtain synthesis and secretion of heterologous proteins in S. cerevisiae.
Use of signal/leader peptides homologous to yeast is known from i.a. U.S. patent specification No. 4,546,082, European published patent applications Nos. 116 201, 123 294, 123 544, 163 529 and 123 289 and DK patent application No. 3614/83.
In EP 123 289 utilization of the S. cerevisiae a-factor precursor is described whereas WO 84/01153 indicates utilization of the S. cerevisiae invertase signal peptide and DK 3614/83 utilization of the S. cerevisiae PH05 signal peptide for secretion of foreign proteins.
U.S. patent specification No. 4,546,082, EP 16 201, 123 294, 123 544 and 163 529 describe processes by which the a-factor signal-leader from S. cerevisiae (MF.alpha.1 or MF.alpha.2) is utilized in the secretion process of expressed heterologous proteins in yeast. By fusing a DNA sequence encoding the S. cerevisiae MF.alpha.1 signal/leader sequence at the 5' end of the gene for the desired protein secretion and processing of the desired protein was demonstrated.
EP 206 783 discloses a system for the secretion of polypeptides from S. cerevisiae using an .alpha.-factor leader sequence which has been truncated to eliminate the four .alpha.-factor units present on the native leader sequence so as to leave the leader peptide itself fused to a heterologous polypeptide via the a-factor processing site LysArgGluAlaGluAla (SEQ ID NO; 76). This construction is indicated to lead to an efficient processing of smaller peptides (less than 50 amino acids). For the secretion and processing of larger polypeptides, the native .alpha.-factor leader sequence has been truncated to leave one or two of the .alpha.-factor units between the leader peptide and the polypeptide.
A number of secreted proteins are routed so as to be exposed to a proteolytic processing system which can cleave the peptide bond at the carboxy end of two consecutive basic amino acids. This enzymatic activity is in S. cerevisiae encoded by the KEX 2 gene (Julius, D. A. et at., Cell 37 (1984b) 1075). Processing of the product by the KEX 2 protease is needed for the secretion of active S. cerevisiae mating factor .alpha.1 (MF.alpha.1 or .alpha.-factor) whereas KEX 2 is not involved in the secretion of active S. cerevisiae mating factor a.
Secretion and correct processing of a polypeptide intended to be secreted is obtained in some cases when culturing a yeast organism which is transformed with a vector constructed as indicated in the references given above. In many cases, however, the level of secretion is very low or there is no secretion, or the proteolytic processing may be incorrect or incomplete. It is therefore the object of the present invention to provide leader peptides which ensure a more efficient expression and/or processing of polypeptides.